AU2015304820A1 - Novel bacterium of bacillus genus and uses thereof - Google Patents

Abstract

The present work relates to a novel microbe belonging to

Description

NOVEL BACTERIUM Of BACILLUS GENES AND USES THEREOF 'fh© present invention relates to a novel bacterium beteit^ingi:*ö'Äel|l«sr family, designated as Baúitím wbttfis ssp. anti-niicrobiai and/orM and plant growth promoting activity, ís0i^l^:.:.tp.(Í:: MenriSiation of extract of the novel microbe exhibiting antinrierobial and/or antifungal, proteolytic·, amyMyttc activities, composition. comprising the novel bacterium and/or extract, method, of inhibiting the growth of pathogenic microbes and/or fbngi by contacting the pathogenic microbes and/or fungi with an effective amount of the novel bacterium and/or an antimicrobial and/or antifungal and plant growth promotion composition andmr agent and use thereof,

BACÍ£GÍ£OLNI> Ö;F:XME INVENTION

The Earth’s atmosphere is known to team with airborne microorganisms, though the high light intensifies, extreme temperature variations, low ebbeentrations of organic matter and scarcity of water, make the environment unsuitable for microbial growth. Biological material may contribute about 20%, 22% and 10% to the total airborne paniculate matter by volume in remote continental, populated continental and remote maritime environments, respectively, Most of them originate from natural sources such m soli, lakes, animals and humans. Moreover, agricultural, practices, health care units and Industrial operations such as sewage treatment, animal rearing, fermentation processes, and food processing plants also emit viable microorganisms into the environment,

Bacteria form a large domain of single-celled, prokaryotic microorganisms. Typically, a few micrometres in length, bacteria have a wide range of shapes, ranging from cocci to rods and spirals. Bacteria are ubiquitous on Earth, growing in soil, acidic hot springs, radioactive waste, •water, and. deep in the Earth’s crust, as well as in orgardc matter and the live bodies of plants and animals. The bacilli are rod-shaped, gram-positive, spornlating* aerobic or facultative anaerobic bacteria. Most bacilli are saprophytes. Each'bacterium creates only one spore, which is resistant to heat, cold, radiation, desiccation, and disinfectants. The bacilli exhibit an array of physiological abilities that allow them to -live In a wide ran ge of habitats, including many extreme habitats such as the desert sands, hot springs, and. Arctic soils. Bacillus species can be thermophilic, psyehrophilk, acidophilus, alkaiiphihc, iudotolerant. or halophilic and are capable • pf :growing atty ario us pH values;, tempérihures, and salt concentratipnSi;

Production of antimicrobial agents seems to fee a general phenomenon for most bacteria, lásese bacteria produce an admirable array of microbial deferred systems, including broad-spectrum classical antibiotics, metabolic by-products such as organic acids, and lytic agents such as lysozyme, in addition, several types of protein exotoxms, and bacteriaoins, which are biologically active peptide moieties with bactericidal mode of action, are also produced. The biological arsenal from, microbes is remarkable in its diversity arid natural abundance.

The search for new antimicrobial agents is a field of utmost importance. The development of resistance to antimicrobial agents is increasing at an alarming rate. Current solutions involve development of a more rational approach to antibiotic use and discovery of new antimicrobials.

Highly relevant patents 1. Novel bacterial strains and methods of controlling fongal pathogens (WO/2ÖÖO/Ö15761),

objectives m the xmmmmN

The objective of the present invention is to provide a novel bacterium exhibiting antimicfobiai and/or antifungal and plant. grp.Ä:pÖtaoiPh'aetiyi^

The objective of the present invention is to isolate & identify an extract of the novel baetermm, wherein the extract displays antimicrobial and/or antifungal plant growth promotion activity.

The objective of tire providean antimicrobial and/or anti fungal and plant growth: promotion composition or agent wherein the composition or the agent comprises the novel baeferium and/orTheextract^ fynoiher oiyeetiye of the present invention is to provide a method of inhibiting the growth of pathogenic mierobes and/or fungi by contacting the pathogenic microbes and/or fungi- with an effective amount of the novel bacterium and/or an antumerobial and/or antifungal and plant growth promotion composition and/or agent whefoin the composition or the agent comprises the novel bacterium and/or die extract of the novel bacterium and/or a mixture of tíre novel bacterium and its extract, ".The. other objective of the pfesent mvmtion is to provide use of die novel bacterium, an antimicrobial and/or antifungal composition or agent wherein the composition or the agent : comprises the novel bacterium and/or the extract of the novel bacterium and/or a mixture of the novel bacterium and its the· growth of pathogenic microbes and/or fungi piÄipAEY·m rm invention Ämaspeefof the present invention Is to provide an isolated, novel bacterium which is useful In producing äxttimierobiui and/or apdfongai metabolites or agents.

One aspect of the Invention.-IS" fo' prop# a novel form of bacterium belonging to

Bacillus species winch is designated as Bacillm subtilis ssp. shriramensis having accession number (MTCC-S674T In pardeutam the novel haeforium disclosed. in the present work is capable of exhibiting thsfinet «ndmierobial and/or annfungaland plant growth promotion property. Äother aspect of the present mvention k to provide a process for the production of an ptifoicrohial and/or antifungal and plant: gfowth promotion composition or agent wherein the composition or the agent comprises Bacillus subtilis ssp.skriramemis {MTCC-5674) and/or the extract of the Bacillus subtil is ssp. shriramemts (MTCCtS674),

There is provided a composition comprising Bacillus mbfilis ssp, shriramensis (MTCC-5674). The composition may further comprise phannaceutieally acceptable exeiptentSj diluents and/or carriers.:

There is provided a composition: containing an extract of Bacillus subtilis ssp, shriramensis (M1CC-5674). There is also provided a composition comprising an aqueous extract of Bacillus subtilis ssp. shriramensis The composition may1 further comprise pharmaceutically acceptabie excipients, diluents and/or carriers,

Ihere ts provided a method for inhibiting the growth of pathogenic microbes and/or fungi by contacting the pathogenic microbes «nd/or fungi with an effective amount of Bacillus subtilis ssp. shriramensis (MTCC-5674) or the extract Bacillw subiilis ssp, shriramensis (MTCC-5674), The Bacillus subtil^ .asp,:' skriramnsis (MTCG-5674) and/or the extract of Bacillus supifhs ssp. shriramensis (MTCC-5674) may optionally contain one or more additional antimicrobial and/or antifungal: and plant gro wth promoting agents.

There is provided in the present myeniidh foe use of Ba&Iim subtilis ssp. shriramensis (MTCC-5674) and/or the extract of the Bacillm submits·, ssp. skniramMsis (MTCC-5674) in the formulation oí an antímicroi?|i^^|bPt/ímlÍfhng^Í and plantgrowth promotion composition or : agent for inhibiting the growth pf pathopnic microbes and/or fungi. DETAILED DESCMIFITÍM OF THE INVENTION :

The present indention provides a no vel microbe 'belonging to Bacillus family designated as Ba0lmmbtilis ;ssp< shrímmemmmé having an accession niunber (MTCC-5674) and a method ef ipodaemg an antimicrobial and/or antifimgal and plant growlfe proinotidn composition, or agent wherein the composition or the agent Comprises 3mtilus subtiiis sap, shriramenm 674) and/or ihe extract of the Bacillus subtiiis ssp* shrimmemis (MTCC~S6?4).

The present Invention also provides a method of:li^Mt^'^e::^^t&amp;genlO''mÍ0rébe$ and/or fungi by ppntsctmg the microbes and/or .fungi with an effective amount of tire novel bacterium, Bacillus subtiiis ssp. shrirabiCHsis composition comprising the novel bacterium or its extract.

The present invention also provides usé of Bacillus subtiiis asp. shrirabi^nsis ^f£CC-$S74), and/or an antimicrobial and/or antifungal and plant growthpromotion composition or agent .Comprising the novel bacterium Bacillus subtiiis ssp, slmmmemis fMT^C-5674) and/or the Of tfaff 30etUu$MtbiUi$ ssp. shriramemis (MTCC~5674) for inhibiting· the pathogenic, microhei and/or fungi

The novel Bacillus: subtiiis .s^ lshritamensis tpffCC«S6?4) may be utili^d for the mass pmduction of antimicrobial and/or antifungal and plant growth promotion composiüon/preparaíion/ageaí by culturing Bacillus subtiiis sisp, shtw^ém&amp;w :%t suitable growth medium under thvomÄe conditions.

Through deep and careful researches, the -inventors have surprisingly found, isolated and CCitoredta novel bacterium, which can produce a novel agent. Through detailed experimental researches, the inventors have also invented a method of producing the said novel agent from the sard hovel Microorganism, .Brief Description

Fig. 1 - Plate showingisolation and purification- of ssp. shrtramensis (MTCC- 5674) (A) Mother etdtute piate shiming bacterial grovvth dong with fungal--mycelium; (B) PUHfiq-atloft éTÉa&amp;ítíw $übtiliré$p.JMfi%timti$is (MTCC-5674) from bacterial eo!ony(s) in (A), Arrow indicies pntaiwe bacterial colony,

Fig, 2 - Clone of one of the purified colonies of Bacillus suhtiMs ssp. shsirmmnsis (M7CC** $674) showing inhibition of growth ofFusarium oxysporum mycelium.

Fig, 3 - Microscopic picture of vegetative bacterial cells oiB&amp;eillm suhtiBs ssp. shriramemis (Ml'CC-5674) along with spores.

Fig, 4 - fíate Showing actively growing colonies of Bacillus subítíis ssp, shriramensis (MTCC* 5674).

Fig, 5 - Rod shaped BacillmsubBiis ssp. shrFmmmis (MTCC*5674) under light microscope. Fig, 6 - Picture showing results of catalase test; (A) Negative control; (B) Positive control and $2):, Μφ0&amp;.· .ssp, shrisamemis (MTQC~$674) showing positive results for catalase activity,:

Fig. 7 - Plate showing:: amylolyiie activity of Bacillus wbtiBs ssp, shriratmnsts (MTG05674) cultureßitrate.

Fig, 8 - Picture showing results of Ö/F (Oxidauon-Fermeniation) test (A) Negative control; (B) ßacittw mbtißs ssp. shrir&amp;memis (ΜΤΟ(Ν$04} showing color change only at the top portion of the medium; (β) Positive control.

Fig. 9 - Picture showing results of Hydrogen: sulphide production test (A) Negative control; (B) Bstättmmhtiiis ssp, :shrirantcnsA:;(MffCC^5674) and (C) Positive control Fig. if) j Picture showing::: results of 8DS*FACB of the concentrated culture filtrate of Bacillus suhiiiis ssp. shrimmemm (M7GC-5674),

Fig, II - Culture plates showing aniimicrobia! and/or antifungal activity displayed by (A) Bacillus^ suMUis ssp. skrirammsls ^171(75674) colony and (B) Bacillus subtilis ssp. mriramensis (M7GC-5674) culture filtrate. Ftmrimt oxysp&amp;mm culture was used as test fungus.

Fig, 12» Picture showing results of MIC assay of antiuncrohial and/or antifungal compound by the tube dilution method, l to C-2: Pictures of Fmrimm oxysppmm spores (observed under light tnicroscope), after incubating in PPB coiitaitsing different concentrations of antimicrobial and/or ahtiiunga] agent I«28 (Dilutions 1:1 to 1:100), €-1 - Spores in antimicrobial and/or antifungal agent (Crude); C-2 - Control (Spores in PDB broth without antimicrobial and/or antifungal agent).

Fig. 13 - Picture showing results of MIC assay of antimicrobial and/or antifungal agent by agar diffusion method. 1 to C-2: Pictures of Fusarium oxysporum mycelium growing on test plates. 1-28 Dilutions of antimicrobial and/or antifungal agent at .1:1 to 1:100 (v/v), Cl - Well containing antimicrobial and/or antifungal agent (Crude); C2 - Control well containing PDB; C3 - Control well containing 70% saturated ammonium sulfate.

Fig. 14 - Picture showing effect of antimicrobial and/or antifungal agent on spores of Aspergillus niger. (A, B &amp; C) Spores oí Aspergillus rtiger showing normal germination in PDB media; (D) Spores of Aspergillus niger failed to germinate in PDB media containing Bacillus subtilis ssp. shriramensis (MTCC-5674) extract. * Fig. 15 - Plate showing antimicrobial and/or antifungal activity of cell lysate against Fusarium oxysporum; (1) Well containing only lysozyme (to check the effect of lysozyme on fungus Fusarium oxysporum ) and (2) Well containing cell lysate of Bacillus subtilis ssp. shriramensis (MTCC-5674).

Fig. 16 - Plate showing assay of antimicrobial and/or antifungal activity of Bacillus subtilis ssp. shriramensis (MTCC-5674) cells/extract against diverse types of plant pathogenic fungal and bacterial species. Á. Fusarium oxysporum, B. Sarocladium oryzae C. Trichoderma viridae D. Colletotrichum capsicii E. Exerohilum turcicum F. Rhizoctonia solanii G. Macrophomina phaseolina H. Xanthomonas oryzae

Fig. 17 - Plate showing results of antimicrobial and/or antifungal activity of Bacillus subtilis ssp; shriramensis (MTCC-5674) extract on germination of rice seeds in presence of Fusarium oxysporum. (A) Rice seed treated with fungus Fusarium oxysporum spores; (B &amp; C) Rice seeds treated with fungus Fusarium oxysporum and Bacillus subtilis ssp. shriramensis (MTCC-5674) extract.

Fig. 18 - Plate showing results of experiments to show absence of pathogenicity of Bacillus subtilis ssp. shriramensis (MTCC-5674) on various plant species. (A) Rice, (B) Cotton, (C) Tobacco, (D) Com, and (E) Tomato.

Fig. 19 - Picture showing results of experiments to show action of Bacillus subtilis ssp. shriramensis (MTCC-5674) as bio-control agent. (A) Tomato plant infected with Rhizoctonia solani (NFCCI-3194) fungus. (B) Tomato plant with Rhizoctonia solani (NFCCI-3194) and Bacillus subtilis ssp. shriramensis (MTCC-5674) and (C) Control tomato plant (without

Rhizoetcmia solani fungus and Bacillus subtilis ssp. shrimmensis (MTCC-5674).

Fig. 20 - Plates showing (I.) Penicilimin malician {NPCCI-1997) fungal colonies and (2) Bacillus subtilis ssp, shrimmensis (MTCC-5674) j?ure colonies.

Fig. 21 - Plate showing corn seeds coated with various formulations of antimicrobial/antifungal agent Bacillus subtilis ssp. shuramemis (MTCC*Sé^4). 1. (Control-1) Seeds treated with the formulation: which has no fungal pathogen and bio-control agent; 2. (Control-2) Seeds treated with the formulation which has no bio-control agent; 3, (Control-3) Seeds treated with the formulation which has commerci#;|lin^ot(le '‘Carbendaziin WP50”·; 4a, Seeds treated with the fotniulation which has Bacillus subtilis ssp. shrimmensis cells (SxlO4 efu); 4b. -Seeds heated with the formulation which has Bacillus subtilis ssp. shrimmensis ceils (SxlO5 cfu); 4c. Seeds treated with the formulation which has Bacillus subtilis ssp+ Mriramemis cells (5x10* cfu); 4d. Seeds treated with the formulation which has Bacillus subtilis ssp. slwiramemis cells (SxlO7 efu) and 5. Seeds treated with the formulation which has only Bacillus subtilis ssp. shrimmensis cells (5xI07cfu).

Fig. 22 - Plate showing results of bio-control activity after 2 weeks of incubation, 1. (Control-!) Seeds treated with the formulation-i which has no fungal pathogen and antifungal agent; 2. (Control-2) Seeds treated with the formulation-2 which has no bio-control agent; 3. (Control-3) Seeds treated with the fonnulation-3 which has commercial “Carbendazim WP50”;· 4a, Seeds treated with the fomiulaüöti-4a which Bacillus subtilis ssp, shrimmensis (5xl04 cfu); 4b. Seeds treated with the form«Mhdn-4b which has Bacillus subtilis :ssp; shriramemis cells (5x1 Ö5 cfii): 4c. Seeds treated with the formuíation-4c which hm Bacillus -subtilis -asp. shrimmensis cells (5x106 eft#; 4d< Seeds treated with the formulaiion-4d which has Bacillus subtilis ssp. shrimmensis cells (SxlO7 cfu). and $< Seeds treated with fee formulation-S which, has only Bacillus subtilis ssp, shrimmensis cells (5x107 cfu).

Pig. 23 - Plate showing results of bio-control activity after 4 weeks of incubation, 1, (Control-1) Seeds treated with the formulation-1 Which has no fungal pathogen and antifungal agent; 2, (Control-2) Seeds treated with the formulation-2 which has no bio-control agent; 3. (Control-3) Seeds treated with the fonnulation-3 which has commercial fungicide “Carbendazhn WPSÖ”; 4a. Seeds treated wife fee foraiuiation-4a which has Bacillus subtilis ssp. shrimmensis (5xl04 cfu); 4b. Seeds treated wife .fee ibrmu!ation-4b which has Bacillus subtilisssp. shrimmemis cells (SxlO5 cfu); 4c, Seeds treated wife fee fdrmuiation-4e which- has Bacillus subtilis ssp. Wriramensis cells (SxlO6 efu); 4d. Seeds treated with fee formulaiion-4d which has Bacillus subtiUs ssp, shriramensis pells (5x1 Ö? du) and 5, Seeds treated with the fomtulation-5 which has only Bacillus stthtilts ,ssp< sfa'immemis cells (5x107 efe). fig. 24 -Assay of aniifengal a^d/ojr afeinwcrobial apiivity ¿gainst a variety of hitman pathogenic fungal species, Ä* B and © - Assay of antifungal and/or antimicrobial activity against Penmtiitm spp, (A) Bmicillium spp. fuitgal colonies .(B) Mycelium and (C) Effect of Bacillus subí His ssp. s hrtmmcnsis (MTCC-S674) culture iltrate. 1¾ E and F -Assay of antifungal and/or antimicrobial activity against AspcrgMm 'β#νι$ (P) Fungal colonies (E) Mycelium and (F) Effect of Bacillus subí ills ssp > skrimmemis (|CFdC*$674);-q'ulture:;:Í®Íh»te. G, H and 1 - Assay of aniifengal and/or antimicrobial activity against Aspergillus niger, (G) Fungal colonies (H) Mycelium and (I) Effect of Bacillus· subtiBs ssp, shriramensis (MTCC-5674) culture nitrate. 1C and E * Assay of antifungal and/or antimicrobial activity against unknown fungus causing skin infection. (J) Fungal colonies (K) Conidia and (L) Effect of Bacillus sublilis ssp. shriramensis (MTCC-5674) culture filtré,·

Fig.25 - Effect of Bacillus sublilis ssp. skrimmmsis (MTCC-5674) formulation on growth and development of Corn. The corn seed heated with formulation containing Bacillus subtilis ssp. shriramensis (MTCC-5674) showed higher growth rate, biomass and grain yield. ISOLATION AND IDENTIEI€AT!ON 0F TÖE NOVEL BACTERI UM .

Tie Inventors^ ^ilecieá sap|>i^ from IB different locations in Hyderabad and Pataacheru- (Telangsha, India) while conducting a study on air ilota. Disposable petri plates containing medía (T3 Medium, Travers, et ob» 1987} were prepared in fbe laboratory and exposed to air at differanLlppatib#, The exposed plates were sealed and incubated at 30° C in lab incubator* in one of the plates exposed to air in Patancheru area* a bacterial colony surrounded by fungal myeefium was observed fFig> 1A), Despite continued incubation, the clearance zone was maintained and growth of Frugal tnycell um remained restricted to the periphery of clearance zone. The nncrporgatiisms from this colony were subjected to purification by using standard rád%0s! öfmicrQbiology ,(í*g. TS)* :'T&amp;8: ihdMdual eoUpibs were tested against a common ñingas MtiSimim oxyspamm (1¾ 2).

Que of the colonies sliowed inhibition of fangal growth and aclearance zone was observed (Fig, 2), Microscopical examination of the bacteria from the colony revealed a rod shaped motile bacterium (Fig, 5). After six dap of incubation in the; culture medium the bacteria produced spores. The colonies of the bacteria were mucoid, raiseds encalar, smooth, and creamy to off-white in color (Fig, 4), and cells showed Variable gram staining (Fig, 5). A rauge of Mochemieat tests including carbohydrate femienfatlon, catalase activity, oxidation-fomentation test* starch hydrolysis, hydrogen salphide production test, oxidase activity test, desoxyc bolate agar test were carried out The results of these tests epnilrmed that the bacteria is catalase positive, possess amylase activity, strongly aerobic, does not produce hydrogen sulfide, oxidase positive and gram variable.

For identification of bacteria t&amp;S DMA sequencing and FAME analysis was carried out. The results of botli studies showed that the bacteria is showing 0.37% difference in, 16S DMA sequence and FAME similarity index of 0,827; with Bacillus subtilis ssp, subiilis and .0.84% difference in 16S DNA sequence and FAME-· similarity index of Ö.749 with Bacillus alraphmisMm, the results suggest that· this bacterium is related' to Bacillus subtilis and Bacillm&amp;irophmm> hat not idéntica! to any Of the catalogued bacterial species in ATCC collection.

Resul&amp;pf i6SdE)NA sequence ^mparison ·=—. ............. "" " .»..« -—... — ..........

. 1 1 1 “ '·"· ·'·· “ -,-1-,-

Results of FAME analysis comparison

isoi|t^ J?aeteti«m isi a isv member of sub-species of the genus Bacillus. According to bfecteriafc η^ΐϊΐβπ^^. '<K>nve^fon, the novel bacteria! species was named as Bacillus subdiis ssp, shritamémteBtkV^cteúim is deposited in the Microbial Type Culture tSoitecrion (MTCC) at IMTECH, Chandigarh, India, The deposition number of this novel species is (MTCC-5674).

Characteristic Features of the Hovel Microbe having aecessumhlcms i tion number IMTCC-S6741 provided by the present·Invention

The bacterium is a rod shaped measuring 2,45x0.88 pm, motile, spore colonies; arc smooth, mucoid, off-white to creamish in early stages bncium· ν\#&amp;Ι#|ί on, pmlonpd incubation, The bacterium transforms into spore as the .nutrients in the medium deplete, normally the process of sporuiation rakes place In 4 days of incubation in 10 ml medium: containing 100 μί of 5x108cells inoculum in a 25x150 mm culture tube at 3D °C and slinking at 200 rpm..

The noyei; bacterium, 8m0u$ subííUi ssp. skimmensis having accession number (MTCC-5674} exhibits antimicrobial and/of antiiungal activity, The extract of the novel bacterium, Baciiius] suh&amp;M ssp, skrtmmemis having accession number (&amp;^<*p-5'd|4) jiv«xhthits:, antimicrobial and/or antifungal activ%, The range of potential applications and uses of the bacterium: are extensive*

The present invention provides a method of producing the antimicrobial andmr antifungal extract front the novel bacterium, Baciílus Mktiíís ssp. shbimwemk having accession number (MT€C.3b74T .Production and Isolation of AorimicroMal and/or Antifungal Agent

Composition of culture medium for the growth of Bacillus subiilis ssp. shrirumensis having, accession number (MTCC-5674) is as follows 1. . Tryptoxie : 032 % (w/v) 2. Tryptose : 0.24 % (w/v) 3. Teast Extract : 0,1.8

4. NaH2P0.i.H20 : 0,044 M

5. Na2HPO4 : 0.062 M 6. MnCia : 0.000 5% (w/V): pH ~6.8 * 1. The medium was -prepared as per the method given ip Annexure -1 (I) and 100 mi aliquots were transferred into 500 ml conical flasks. The media was sterilised by autoclaving at I2i°G ib.r 15 min. 2: Each flask was inoculated with a single pure colony of Bacillus subiilis ssp, shnmmcmis fMTCC-%5674) and incubated at 30°C, 200 rpm tor 60 hours.

Isolation of antimicrobial and/or anflfunqal agent from culture medium

Following the growth of bacteria in T3 broth for 60 hours, the culture was centrifuged at 12000 tpm for 10 min at 4 °C. The supernatant was collected and filtered using 0,22 urn disc filter (Miliipore/Sartorius). The fíltrate was preserved under appropriate storage conditions for detailed experiments to study antimicrobial tut#» antifungal activity,

The present invention particularly provides a novel microorganism. Bacillus subtitis ssp. shriratmnsis having accession number (MTCC-5674) áí)d a method for the production of antimicrobial and/or antifungal composition from the novel bacterium and/or its extract or a mixture of the novel bacterium and/or its extract.

One embodiment of the present invention provides an isolated novel bacterium- belonging to Bacillus subiilis ssp. shriramensis exhibiting antimicrobial and/or antifungal activity, having accession number (MTCC-5674), in one embodiment of the present invention is provided the novel bacterium designated as Bacillus subiilis ssp. shriramensisMvmg accession numberlMFGG-5674),

In another embodiment of the present invention there is provided a pure erdture of The novel bacterium. Bacillus subrtlls ssp. shrir&amp;mefflk having accession number (MTCC-5674), in one embodiment ox the present invention there is provided ap: extract of the novel bacterium, Bacillus subUUs ssp. shriramemis having accession number (MTCJC-5674) wherein the extract exhibits antimicrobial and/or antifungal.activity, in. mipther embodiment of the present invention there is provided an extract of the novel bacterium, BaciUmmibtU^\ss^cshrinmemis:havihgSession number (MTCOS674) wherein the extract exhibiting antimicrobial and/or antifungal activity is an aqueous extract. In yet another embodiment of the present invention diere is provided a process for the production of the extract of the novel bacterium, Bacillus mbiiiis ssp. shriramemis having accession number (MTCC~567$f:. where in the process cotnpflses growing the novel 'bacterium, Bacillus subdlis ssp, shriramemis having accession ntúnber (3VÍTGC-5674) Is a nutrient medium and recovering the extract haying antifungal activhy by using convendoxial metiiods.

In another embodiment of the pxcsetli invention there is provided a process for the production of the extract of the novel bacterium, ΒφΒΒ®. subtilte ssp, shrimmenMs having accession number (MTCC-5674} wherein the process comprises growing the novel bacterium, Bacillus subtiHs ¡ssp, shriramemis having accession number (MTC€-Sb74) undenacrobic conditions.

In yet aiiother embodiment of the present invention diere is provided a process for tike production of the extract of the novel bacterium, Bacillus sublUls ssp. shriramnsls -having accession number £MTC£>e>674> wherein the process comprises growing the novel bacterium. Bacillus subii-Us ssp. sheßrämemis in a nutrient medium, recovering the extract .having andplcrobia! and/or antifungal activity and optionatly comprises concentrating the extract: using conventional methods.

In one embodiment of the present invention, there is provided a composition comprising the novel bacterium. Bacillus subtilis ssp, shriramemis having accession number (MTCC-5674) wlierein; the coxhphá.Uo» h^sprimio^bial and/or antifungal actívity,

Ih another embodfmeni of the present invention, diere is provided a composition comprising the extract of the novel bactemijpa, Baclllm subtilis ssp, shriramemis having accession number (MTCC-5674) whet ein the composition has antimicrobial and/or antifungal activity.

In another embodiment pf the present invention, there is provided a composition comprising the •novel bacterium, Bacillus subtilis ssp, shrimmensis Having accession number (MTCC-5674) and the extract of the.novel bacterium, Bacillus subtilis ssp. shriramemis having accession number (MTCC-5674) wherein the cQm^^riomte\andmicrobial:,aid/or^dfungal -activity. in one embodiment of the present invention, there is provided a composition comprising the novel bacterium. Bacillus subtilis ssp, shriramemis having accession number (MTCC-5674) and/or an extract of the said novel bacterium, Bmffltis subtilis ssp, shriramemis, or a combination thereof that optionally comprises one or inore antimicrobial and/or antifungal agents. in another embodiment of the present invention,, there is pra^^^-ä/^iöposition:comprising an extract of the novel bactpritim*.: Bacillus .subtflfs '®sp* "'sfmkmma^s having accession number (MTCC-5674) that optionally comprises one or more antinrierobial and/or- antifungal agents, |n yet another embodiment of the present invention there is provided a composition comprising the combination of the novel bfpteriipi»0αφψ shriramemis having accession; number (MTCC-5674) and its·· "tilgt. ^comprises one or more antimicrobial and/or antifungal agents.

In one embodiment; of the present invention there is provided a composition comprising the -novel bacterium, Bacillus-subtilis ssp, shriramemis liming, accession number (MTCC-5674) or an extract of the novel baeteri\m,Bmillm subtilis ssp. slmimmemis. having accession number (MTCC-5674) or a combination thereof that optionally comprises agriculturally or pharmaceutically acceptable carrier.

In another embodiment of thepresent invention there is provided a composition comprising the novel bacterium,'Bacillus subtilis: ssp, shrirnmetisis having accession -number (MTCC-5674) that optionally comprises agrieulíp^íy orpharitmceutically acceptable carrier.

In yetanother; einbodirnent^of die pre®nt: invention there is provided a composition containing an extract of the novel bacterium, Bacillus subtilis ssp:. shriramensis having accession number (MTCC-5674) that Optional ly comprises agriculturally or pharmaceutically acceptable carrier.

In still another embodiment of the present Invention there is provided a composition comprising the combination of the novel bacterium, Bacillus stibtilis ssp. shriramemis having accession ¡ßpB^i^TCC-56^) and an extract of die said novel bacterium, Bacillus subtilis ssp,' s&amp;imméwist:-y^ch. optioml£;cbmprises ag^óúltutáíly iic<^!y>le.<^^er:<See:j:^^exm;IH), &amp; one embodiment of the present invention them is provided a method for-inhibiting· growth of pathogenic fungi and/or bacfigááf wherein :.sdd method -compaséis contacting the pathogenic fungi and/or bacteria with an pffectiye amount of the novel bacterium, Bacillus subtilis ssp. shrimmensis having, accession luxniber {MTCC~S674) or a composition comprising the said novel bacterium, or its extractor a combination ihereori

In one ^^diment/Qf;||f ibsm is provided a method for inhibiting growth of pathogenic ñmgi and/or bacteria, wherein said method comprises contacting the pathogenic fungi and/or bacteria Mth an effective amount of the novel bacterium, Bacillus subtilis ssp, shriramcmis ha ving accession number (MT€€~SP74}<

In another embodiment of the present invention there is provided a method for udiibiting growth of pathogenic fungi and/or. bacteria, wherein said method comprises contacting the pathogenic fungi and/or bacteria with an; effective amount of a composition comprising the novel bacterium, jBaciUuS:Sub0}i$ ssp. accedió» number (MlfcC*5674),.

In yet another embodiment of the present invention there is provided a method for inhibiting growth: of pathogenic fungi and/or bacteria, wherein said method comprises contacting the pathogenic lung! and/or bacteria with an effective amount of a composition comprising an extract of the novel baeBacillus subtilis ssp, sbriramensis (MTCG-5674) wherein the extract has activity.

In yet another embodiment of the present invention there is provided a method for inhibiting growth of pathogenic fungi and/or bacteria, svherein said method comprises contacting the pathogenic fungi and/or bacteria widt an elective amount pi a composition comprising the novel bacterium, Bacillus subtilis- ssp, jhrimtmtpis (MTCC-fÖ74) and an extract of the said novel bacterium, Bacillus subtilis ssp. shtirntnensiS/ wherein the extract has antimicrobial and/or antifungal activity.

In one embodiment of the present invention Itere is provided a use of the novel bacterium, Macilim suBälisMp' shrimmnsis having accession number (MTCC-5P74} or a composition : comprising the said novel bacterium or its extract or a combination thereof for tire preparation of an antimicrobial and/pr antifongal composition for inhibiting the growth of ; pathogenic iungi and/or bacteria, in another embodiment of the present invention there is provided a nse of the novel bacterium, Bacillus subtilis ςψ. sk’tmmmsis having accession number (MTCC-5674) for the preparation of an anti microbial and/or antifungal composition for inhibiting the growth of pathogenic fungi and/or bacteria.

In another embodiment of the present invention there is provided a use of the composition comprising1 the novel: bacterium,, Bacillus subtilis ssp, shrirpmensis (MTCC-5674) for die preparation of an antííperobial -apd/or -aptifongal composition for inhibiting the giowth of pathogphiC'^ngi^/Öf'bacteria.,

In another embodiment of the present invention there is provided a use of the composition comprising an extract of the novel bacterium, Bacillus suMBs ssp, shriramensis (MTCC-5674) for the preparation of an antimicrobial and/or arfofungal composition for mhibiting the growth of pathogenic fungi and/or bacteria.

In. another embodiment of the present invention there is provided a use of the composition comprising the extract of tl® novel bacterium, Bacillus subtilis $sp> shriramensis (MTCC-5674) and an extract of the said novel bacterium, Bacillus subtilis ssp, shriramensis. for the preparation of an antimicrobial and/or antifungal composition for inhibiting die growth of pathogenic fungi and/or bacteria, in another Cmbodimemc there is provided a pharmaceutical mid agriculturally effective oamposhion comparing the novel bacterium,, Bacillus subtilis ssp, shriramensis, having accession number (MTCC^S674:).

In another embodiment, them is provided a pharmaceutical and agriculturally effective composition comprising the extraet of the novel bacterium, Bacillus subtilis ssp. shriramensis (MTCCfSd? 4).

In yet another embodiment of the present invention there is provided a method of producing the said effective composhipnfroni die novel bacterium. Bacillus subtilis ssp. shriramensis having accession number (MTQC*5674·).

In yet another embodiment of .the present ihyemion, the steps and time required for the production of the said, composition/extract are kept at die minimum duration coupled with the : maximum recovery of the compound.

Other advantages or benefits of the present Invehtion

The bacterium Bacillus suhtUis ssp. shriramemh'{MTCC-5674) along with antimicrobial and/or antiftmgal agent also produces strong thermophilicprotease: and amylase which are active even ; after exposure to high temperature i.e., 12l°C for I Smin.

The present invention is further explained by the following examples. However, the present invention Is not limited to these examples in any manner. The following examples is intended to •illustrate the working of disclosure and not intend:ed to take restrictiveiy to apply any limitations on the scope of the present .ϊην&amp;ηΐίο#..Ί^ρ$β' perno«« skfiled in the art will understand that tire equivalent substitutes to the specific substances described herein, or the corresponding impmvements are considered to be within the scope oTthe mveppon.

Detailed Methodology is explained lit the following examples;

The methods· employed in the present work are well-known in microbiology with the respective parameters varied and optimized fqr the present study.

Example 1 1.1 Collection and preliminary screening of air samples

Air sampling was carried out at different locations in Hyderabad and Patanchem (Telangana, India), Disposable Petri plates containing T3 medium were prepared in the laboratory and exposed to air at different locations. The exposed plates were sealed and incubated at 30°C in lab incubator. In one of the plates exposed to aif in bacterial colony surrounded by tungal mycelium was observed, 1.2 Preliminary screening of air samples for antimicrobial and /or antifungal activity

Despite continued incubation the clearance zone was maintained and growth of fungal mycelium remained restricted to the periphery of /clearance zone. The microorganisms from this colony were subjected to purification by using standai'd methods of microbiology (Fig. IB). The individual colonies were tested against a common fungus Fusarium oxysporum (Fig. 2). One of the colonies showed inhibition of fungal growth .and a clearance zone was observed (Fig. 2). 1 -3 S^ningj^fno^iJsoíge

Evaluation of the bacteria under imeroseope revealed that it is a rod shaped motile bacterium (Fig, 5). After six days of incubation the bacteria produced spores,

The colonies of the bacteria: wep ,|rp^t4.;#sé4 circular, ¡smooth, and creamish to off-white in color-(Fig, 4|:dndthe cells showed variable pam staining.

Example 2 2.1 Characteri 2.1.1 €teMéterl^tíg.atifthe::nOvei IsolatzBmíUmmbiiiis ssp, shrirammls havlnsf ; accession number (MJCC-SCä) A range of luoehemíeai tests, including carbohydrate iertnentation, catalase test, -oxidation-· fermentation test, starch hydrolysis, hydrogen sulfide production test, oxidase activity test, were carried out, The results of tírese tests confirmed that the bacterium is catalase jrosi tive , amylase positive, oxydase positive and strongly aerobic, : 2,1,1.1 IMTC^^

Colonies of Badilas m&amp;iilis ssp, dirirarnmsis .(MTCC-5674) are mucoid, raised, q^ö|ars..:SteOöth,.ötiddreattd^tö ofMáte in color (Fig. 4), 2,1,1.-2 .C.ultum.,Charact^ds.tigs

Bacillus subtUis ssp, shrimtmmls :(MTCC~5074) shows optimum .growth at 30"C (can grow from I5'C to 55"C). As It is an aerobic bacterium, it requires adequate oxygen for its growth, needs continuous shaking for culturing in -broth. I 2.1,1.3 „Cd!.morphology

Badlius stibtilis ssp. shrirmensis (MTCC-5674) cells are rod shaped, diplobacilh and motile (Fig, S). 2,1,1,3,1 Comparison of colony growth and morphology of BacMlm subtilis ssp, shriramemis (MTCC-5674) with that of Badlius subtilis and Badlius atrophaem

2.1.1.4 Catalase test

Material • Culture tubes of Bacillus subtilis ssp. shriramensis (MTGC-5674) • Hydrogen Peroxide Method • Three tubes containing LB medium were labelled as “test”, “positive control" and “negative control” a loop full of Bacillus subtilis ssp. shriramensis (MTCC-5674), Escherichia cotí and Streptococcus pneumonia were inoculated in the tubes respectively. Following incubation at 30°C for 24 hours, few drops of hydrogen peroxide were added in all the tubes and observed for formation of bubbles.

Result

Gas bubbles were formed both in “test” and “positive control” tubes indicating that the Bacillus subtilis ssp. shriramensis (MTCC-5674) is catalase positive (Fig. 6). 2.1.1.5 Starch Hydrolysis Material • Bacillus subtilis ssp. shriramensis (MTCC-5674) culture filtrate • Starch agar plates • Iodine • Incubator Method

The starch agar medium was prepared as per the method provided in the Annexure - I (VI). Two wells were made at equal distances in the plate containing starch agar medium and labelled as “test” and “negative control”. An aliquot of 500 μΐ each of Bacillus subtilis ssp. shriramensis (MTCC-5674) culture fíltrate and sterile distilled water were dispensed into the wells labelled “test” and “negative control”. The plate was incubated at 50°C for 4 hours.

Result

After 4 hours of incubation, th«.'Blue color surrounding the test; weft disappeared Indicating that the Bmilhs suhfilis ssp, shrimmemis (MTÖP-5674} culture Mírate has amjdo||tie: activity. No change In the blue color was observed mthdarea.sivnx5imd^::ediitlÖl well (Fig, 7)...

Material * l|ugsh Leftsotlls OF Basal Medium « Testfufoes * M-coIlmliure * sfoimmémis (M%£CS4$$}.· * Incubator Method

Three tubes containing Hugsh Iwiison’s OF basal medium (OFBM) (Annexure - I: (YII)) were labelled as “negative control” “positive control” and ¥e$f! and a loop full of Alcaiigmes ßemiis, EscHericnm mli mä Bmßm snhtßs ssp, ßrimmenm (MTßC-5674) was inoculated in the tubes respectively. The ttihes were incubated at 30CC for 48 hours and observed for ehange; of color.

Result it has been concluded from the observations that the test organism (Bacillus svhfííp issp, shrPaméMls (MTCC-5674) is strictly aerobic as it did not ferment carbohydrate (neither gas formation nor the color change) deep inside the inedlum, Due to avaiiability of" oxygen on the surface of medium some color change was observed, Whereas E: voll grew very well deep inside the medium and fermented the carbohycfrates fboth gas formation and change ih colör of the medium} indicating that it is a facultative anaerobe (Fig. 8). In the negative control neither gas formation nor color change vus observed,

Material * SIM (Sulilde indole Motility) medium * Culture tubes: " » Rcoi i culture * BáM^:-0$^lmMp^s-HrirmmHsfs'^TCCrrí67'4} ♦ Incubator Method « The tabes eoafeining SIM (Sulfide Indole Motility, (Annexure ~ I (VII)}] .medium were labelled as ^negative .control* and “test” and a loop full, of B. mil and Bmiilus stíbtíim ssp, shrimmemis (MTCC-5674), were inoculated in the tubes respectively, and incubated at: 30°C far hours and observed for color change.

Result

Front the observations, it has been concluded that the test organism is negative For %S production as the medium did not turn black. The same msulf was observed in the negative control (Fig. 9). 2.1.1.8 Effect of pH on the growth of Bacillus subtiiw ssp. shrimmemis t'MTCG-5674)

Ctiliure tubes containing standard culture medium (LB) adjusted to different pH values rtetging from 3,4 to 1LÖ (acidic to basic) were used to grow BmMus $spt Shipifámemís (MTCG-3674) Under standard conditions.

Growth of BacMiut mbtiiU ssp, shrimmmsis (MTCC-5674) was observed in a pH range of 6.4 to 7,2: and the optimum pU was found to be 7.Ö. 2.1.1,9 Antibiotic Sensitivity Test of Bacillus sitbiilis ssp. shrtramemLs HvlTCC- 5674) A 24 bourn old BamBus siihtiim mp. shrinmemis (MTCC-5674) culture was spread over the surface of Ϊ3 agar. Different antibiotic discs were placed on the surface of the T3 agar plates labelled with the respective antibiotic. The plates were incubated at 30*C For 24 hours.

Table-1 Observations on antibiotic sensitivity of Bacillus mbtilis ssd, shrímmensis ÍMTCC-5674) RES - Resistant INI' - intermediate. SEN -Sensitive

Result

1 has been conciuded from th© observations that Bacillus subtilis ssp, shrimmemis (MTCC-5674) is resistant to the antibiotics - ampicÜÜn, carbemeiliin, kanaraycin, enroioxacin, lincomycin, amopcallin, clindamycin, neomycin, azithromycin. The test bacterium is sensitive to gentamicin, tobramicin, vancomicm, novobiocin, suifisoxazole, cephaiothin, chloramphenicol, eeftflaKone and baeipaem and showed intermediate" iesistanee^O;OxaciUm>.am|^>in, streptomycin und erythromycin,. .2-1-2

For ideniifíepon of bacteria, IPS ®MA sequencing and FAME analysis were carried out- Thef esdtS: of bacteria is showing 0.3/% difference in t6f:;^A^:se^ue»ce.':^d B^ME similarity index; of 0.827; with Bacillus subtHis ssp. subiilis an#!.%B4^':scBÍ%et«S8 |n Í6S DMA sequence and FAME similarity index of 0,749' with Bacillus airaphacus, Thus, the resuits indicate that this bacterium is related to BaciUm suhiiiis mid BaciMm airophmust but trot identical to any of the catalogued bacterial species in ATCC collection. 2|E2,i Results.ofi^..DNA^

2d.2,2 Eesults

The isolated bacterium is a new member of the genus BaciUm. According to bacteria! nomenolatitre convention* the hovel bacterial, species was named as Bcmi'us subiilk asp. sbmrmmmm 71¾ bacterium is deposited in the Microbial-.. IVpe Culture Collection ^ IMIECH, Chandigarh, India, The deposition ; number of tins novel species is (MTCC~S674).

Examples %i ..gjááygSáajand^sia£Eiaiaaua£xhsLjgd^lmi^JbiaLíaffl^ 1.1 -1 ProducUQn..Qf.atnimiero.bkLaml/ar..ataiñiftgal. agent Material * T3 broth -1 L : • /ConicaFFlask - 2-ίΕ:-οΐρης%' e Kanamycin (30 pg/ral) * Bacillus suhtiiis ssp, skrteeme^$:Q&amp;TG&amp;?$634) ;S.hakmg,'iftcubatpr (set ab temperst 'Sc 2Qi;:tpm-shökmg)'

Method ' iÄg"T3 broth was prepared according the method described in Annexure - Ϊ (1), A .1 fed culture WB&amp;tii&amp;ts 'mhffl&amp;ssp. sh^ammsiSy-^XCC*5674) was inoculated into the sterile *p. m :the sfeskitlg Incubator at 30°C for 60 hours, while /Shaking at 200 rpm, Followed by the: growth of Bacillus suhtiiis ssp, shriramemb :|ΜΓ€€τ:5;6?^) in the T3 broth for 60 honi'Sj the culture medium was centrifuged at 12000 rpm and 4°C for 10 mih, The supernatant was collected and passed through 0,22 prh lilters to separate put ahy residual bacterial cells. The filtrate was maintained at 3,1.2 Screening the antimicrobial and/or afoifoneal activity of the culture filtrate collected l In step 3.1.1 above i Material ; * Culture Filtrate of Bacillus subiiiis ssp, shriranwmis (MTCC-5674) containing { antirnicrobiai and/or antifungal agent > : i PDA plates » [ Test fungus Fusarium oxyspomm ® Incubator Method : To rest ihe activity of antimicrobial and/or ahtlfihrgai agent in die filtrate, a well was I made in one comer of the PDA agar plate, 500 μΐ of the filtrate was placed in the I well. A loop full of the fungus Fusarium myspartím was inoculated at the other i corner in the same PDA agar piale and incubated for 5 days at room temperature, : Inhibitory activities of the filtrate against the fangps Fiismium oxyspomm were recorded as the inhibitory zone^ strffoundmg the wellin millimetres'.

Result

Clear inhibitory zone of 14 mm was observed surrounding the- well, suggesting that : the; method used for the production of antimicrobial and/or antifungal agent is Optimum..'. ' 3.2 Characterization oí antinÚMolM aoeat

The tmffoiicrobial and/or antifungal activity associated with Bacillus subtilis ssp, skiramemis was invesíigate4i:tO'ascgftaití thé nature of agent causing antimicrobial and/or antifungal aetlyriy.

Material

* T3 broth — 1 L * Conical Flask - 2L capacity

* Kanipyeln (3G gg/raO * Baeüiw subtilis ssp* shnramemis (MTCC-3674) inoculum * ShalStng Incubator MMisd

The T3 broth was prepared according the method described in Anncxure - Ϊ (1), A 1 ml aliquot of 24 hours old Bacillus subtilis ssp, shirammsis (MTCC-5674) was inoculated into feér sterile T3 broth and incubated in the shaking incubator at 30°C for 60 hours, while shaking at 200 rpra. Following growth of BaciUus subtilis ssp, shriramensis (MTCC-5674) in the T3 broth for 60 hours* the culture medium was centrifuged at 12000 rpm and 4°C lor 10::115¾ The supernatant was collected anti passed through 0,22 μιη· filters to remove any remaining bacterial cells. 3,2>i : fMICC-.3674) bacterial cells i To test the activity of antirni^bM^I/orandluagal. agent-by the ceils, a loop foil of s Bacillus subiilis ssp, shrimmemis (MTC£T-5674) Inoculated in one corner of the T3 ; agar plate and a loop fell of the fungus Fmwmm axysporum was· inoculated at the other corner in the same Tf: agar plate and incubated for· 5 days at room temperature, i Inhibitory activities of the bi^te?ial-c^:-aiain^4hp:'-fttugus Fusarium axysporum were recorded as the inhibitory aone surroundingsthe bacterial colony in millimetres,

Midi

Clear inhibitory zone of 14 mm (Fig, 11 A) was observed surrounding the bacterial colony, suggesting that the active compound secreted by the bacterial cells is getting diffused out In the culture medium resulting in clearance zone away from bacterial colony. 3.2.2 Antimicrobial and/or antifungal assay with the Bacillus subtilis ssp. shriramensis (MTCC-5674^1 culture filtrate

To test the nature of antimicrobial and/or antifungal agent in the filtrate, a well was made in the PDA agar plate and 500 μΐ of the filtrate was placed in the well. A loop full of the fungus Fusarium oxysporum was inoculated at the diagonally opposite end of the same PDA agar plate and incubated for 5 days at room temperature. Inhibitory activities of the filtrate against the fungus Fusarium oxysporum were recorded as the inhibitory zone surrounding the well in millimetres.

Result

Clear inhibitory zone of 14 mm (Fig. 1 IB) was observed surrounding the well, suggesting that the filtrate retained antimicrobial and/or antifungal activity, thus indicating the active compound is secreted outside the bacterial cell in to the culture medium. 3.3 Determination of MIC of Bacillus subtilis ssp. shriramensis (MTCC-5674V antimicrobial and/or antifungal agent 3.3.1 Lvophilization of antimicrobial and/or antifungal agent Material • Culture filtrate of Bacillus subtilis ssp. shriramensis (MTCC-5674) • Ammonium sulphate • Freeze drier Method

The antimicrobial and/or antifungal agent was produced and purified by the methods explained in 3.1.1. A 800 ml aliquot culture filtrate was mixed with 382.18 g ammonium sulphate at 70% (w/v) saturation (modified protocol of Jing et al.f 2009) and solution was gently mixed by stirring for overnight at 4°C. The suspension was centrifuged at 10,000 rpm for 10 min at 4°C. The pellet thus obtained was lyophilized for 24 hours in a freeze drier and the dried pellet was stored at room temperature. 3.3.2 MIC of Bacillus subtilis ssp. shriramensis (MTCC-5674^ antimicrobial and/or antifungal agent

Methods 3.3.2.1 Tube dilution m#töd 3.3.2.2 Agar diffusion method * Preparation of slock soiuüpnMlyonMi^ and/or antifungal agent

The stock solution was prepared by dissolving 1 g of lyophilized powder of : antimicrobial and/or.-ac^ftingal agent in SO si! phosphate buffer (pH 7.0), The final : concentration of the stock solution was adjusted to 20 pg/pi. This stock solution was used for making dilutions withPBimedia in different ratios as shown in Table-1. 3,3,2.1 'lube dilution method Material * 1.5 ml tubes * PDB medium * Antimicrobial apd/or autlfuitpi agent stock » Fu$ariiM,myspawM-%me s^pehstött

Method MIC assay of ammdemhiul and/or antifungal agent was carried out in 1.5 ml tubes. Different dilutions of antiiniörobial apd/or anüfUaga! agent were prepared in PDB medium (Table *2) ranging from 10 pg/μΐ (1:1) to .198 ng/μΐ {ítiÓP). pssay wa| parried out «gainst .Pmariim ozysparum by adding 30 μΐ (5χ10δ eiii/ml) of spore suspension in all the tubes and were incubated at 28-0,Jot 2 days, shaking at !80 t|un.

Three contÉ^^^T^á^pne with undiluted antimicrobial and/or antifungal agent stock, second with 70% ammonium sulfate in PDB and third with only PDB. Medium m-:alTtbe:;three: tubes was inoculated with 30 ul (5x10* cfu/ttd) of i^sarhmi üxyspomm spúm^ smg^mmn änd wcrQ incubated M 2B0Cf for 2 days, while shaking at ilÉipm

Material « PDA (Potato Dextrose Agar) plates « PDB (Potato Dextrose Broth) medium • Antimicrobial and/or antifungal agent stock * Fusarium p^sportm

Method MID assay of lyophilked Bacillus subtBis ssp, shrimmensh (MTCC-5674) aniimterolÄ agent was also carried out by agar diffusion method. Four wells of 9 11m diameter each were made at equal distances in PDA plates, Diilerent dilutions of antimicrobial and/or antifungal agent were prepared in PDB (Table-3) ranging from 10 pg (1:1) to 198 ng (1:100). An ηΙΙρ,οί 2ÖÖ pi of each dilution place in to the well labeled with the respective diiudom llie test fungus Fumiwftt oxysponm w>as inoculated in the center of the PDA medium and the plates were incubated at-28¾ for 4 days, A plate with three controls, one containing undiluted antimicrobial and/or antifungal agent stock, the second containing only PDB broth and the thud containing PDB with 70 % ammonium sulphate seas used as control. The activity was measured as inhiMtory mne in millimeterssmiTounding the well. Result

Tube dilution method (Fig, 12)

The samples were observed after 48 hours of incubation under light microscope for spore germination. Spores did not germinate in the tubes containing anümterobialand/or antlhtógal agent in the ratios 1:1,1:2, 1:3 and 1:4. Moderate spore germination was observed in the tubes containing antimicrobial and/or antífUítgal Igdritvín the ratios 1:5, 1:6, 1:7, 1:8 and 1;9 dilutions, and normal Spore germination and mycelia formation was observed in the remaining tubes confining the antimicrobial and/or antifungal agent in the ratios 1:10 to 1:1 ÖÖ (table t).

Tab!e>2 MIC of antimicrobial and/or antifungal agent by tube dilution method

Aaar diffusion method (Fig, 13s)

Inhibition .-of rhngal mycelium growth was· observed around the wells containing antimicrobial and/or antifungal agent in the ratios 1:1,1:2, 1:3 anil 1:4 (Table 3). Moderate inhibition was observed surrounding the wells containing and in icrobial and/or antifungal agent in the ratios 1:5, 1:6 and i:7 dilutions arid no inhibition were observed in the remaining dilutions (from 1:8 to 1:130) (Table 3}. .

Conclusion firorn the above experiment it is conchided that the antimicrobial and/or antifungal agent in powder of crude extract is inhibiting spore germination as well as mycelium growth apto a dilution of 1:4 (v/v), in a concentration dependent manner-

Tabíe-3 MIC* ageiit'by.^M4í%siQ^.RÍ|tli^.

3.4 To test the antimicrobial and/orairtTunRaL^^^^ mMUzßs&amp;MrMmHmk ,(MTC.Cr.Sil# - * PDA agar piales * LB Broth

BmiHus suhtiíls ssp. shriramensflf * Lysozyme * Incubator

Methods ·.<·· 3,4¿ 1 Bacillus subtills ssp. shrirmwsis (MTCQ-5674) egliims

The LB broth was prepared uecortfing to the method described in Annexure ··· i (3). A single colony of Bacillus sufäiUs ssp. sfmmnemis .(MTCC-5674) was inoculated into the sterile LB broth and hsct$>ate$ .at '$Be€:'£br 24 hours. After 24 hours of tncohaiion the vegetative cells were eotieeted by eeatrifligatlón at 6500 rpm, 4°€, tiie ceils were washed thrice with sterile distilled water Md subjected to cell lysis by incubation in .lysozyme at 37°C for 2 h. After lysis the suspension was centrifuged at 1.0,000 rpm for 10 nun at 4*0 to remove the cell debris. The supernatant was collected and passed through 0.22 pm filter and stored at # C. 3-4.2 Antimicrobial and/orjsnqfprg4^^lte#.lpi^'

Two wells were bored in two diagonal ends of the PDA agar in -the petrl plate and labelled one as “test'* and the other as ίΤ·οη0θΓ,< An aliquot of 500 μΐ of the lysate was added to the test we.U and SOO gl of only lysozyme was added to the control well. The test fungus Fusarium oxyspomm was inoculated in the middle of the PDA agar and .i · Incubated for 5 : &amp;g§aU

The cell lysate did not exMbh: aplimierpbkl and/or antifungal activity (Pig, 15} against the fungus Fusarium oxyspamm, suggesting that the antimicrobial and/or antifungal agent is primarily secreted out into temedla* Ésampted 4.1 To test', the.antimicrobial and/or anttftingal; activity aaalnsf other pathogenic fungi

Material * Bacillus subtUis ssp> shrirawpmis (MittXFS$74) * Í%ihdg£híc fungí : 1. Rhizoctonia sofoef (Causes sheaife blight in members of family soianacbh), .2." Samciadium <C^@s:^^h,íOtitt.ríce) 3 . Colletotrichum capsicii (Causes antliraenose in chilli). ;4, ExerokUum iurclcum (Canses tureicnm blight), 5, Macrophomina phaseoiina (Causes charcoal rot). * 13-toth * T3f~ Agar plates * iPIM ··· Agar plates * incubator Method 4M To test the antimicrobialaitd/or antlfeagaf activity mÉi Bmillusmhiilis^ attains! various plant pathoncnic inno.i A loop full oí Bacillus äirinrnmm^:.:$MCC-4674) cells and. a loop Ml·' . of test fungi were inoculated at the diagonally opposite ends of the T3 plates labeled with the respective fungus and incubated at; 28°C till the growth of fungal mycelium was observed in the vicinity of the baeterial colony. 4.1.2 To test the aniimierobial .andfea^iií^^^ giant pathogenic .tunsi' .An aliquot of 5Q0 pi of the cdi^-r^lÄf-eohbttning antimicrobial and/or antifungal agent was added into die welts made in the PDA agar and a loop full of test fungi were inoculated at pope? of the respective plates labeled with the j^spective fungus and meubamd at 28!?£ till me growtli of fitn^rmycelium was; observed in the Vichuty of die welt containing culture fíltrate.

The inhibitory activity of the filtrate against the taipt fungus was recorded in millimetres as the inhibitory zone foime^

Essali A range of fungal species causing diñases in plants were tested in the antimicrobial and% antifungal assay and all of them demonstrated complete inhibition of growth in the presence of JSacUim sithtiiis! ssp, shrirhmensk (M1CC-5674) cells and ¿uso its cultpre filtrate. 4.2 Efficacy of antimicrobial and/or antifungal agent in protecting rice seed from fungal attack Rice seeds were treated with Fusarium oxysporum spores and Bacillus subtilis ssp. shriramensis (MTCC-5674) culture fíltrate and placed in the petri plates containing plain agar to check the efficacy of Bacillus subtilis ssp. shriramensis (MTCC-5674) antimicrobial and/or antifungal agent in inhibiting the fungal attack on germinating seed.

Control seeds were treated only with Fusarium oxysporum fungal spores.

Result

In presence of Bacillus subtilis ssp. shriramensis (MTCC-5674) antimicrobial and/or antifungal agent fungus failed to infect the seeds and the rice seeds germinated normally. However, the seeds treated only with fungus showed severe infection and failed to germinate (Fig. 17), 4.3 To test the pathogenic nature of Bacillus subtilis ssp. shriramensis (MTCC-5674’) of plants

Material • Bacillus subtilis ssp. shriramensis (MTCC-5674) in 1 % CMC in sprayable form • Rice, cotton, tobacco, com and tomato plants • Sprayer Method

The Bacillus subtilis ssp. shriramensis (MTCC-5674) culture was extensively tested for pathogenic behaviour if any, on a range of plant species.

Bacillus subtilis ssp. shriramensis (MTCC-5674) was inoculated into 1 L sterile LB broth in a 2 L conical flask and incubated at 30°C, for 24 hours, shaking at 200 rpm. Following the growth of the bacteria, the cells were harvested by centrifuging at 6,500 rpm, at 4°C for 10 min. The pellet was washed twice in phosphate buffer (pH 7.0) and made into slurry in 1% CMC (Carboxy Methyl Cellulose) in phosphate buffer (pH 7.0). This suspension was used for spraying on crop plants like rice, tobacco, com, tomato and cotton.

Result

From the observations it has been concluded that, all the plant species (rice, tobacco, com, tomato and cotton) sprayed with Bacillus subtilis ssp. shriramensis (MTCC-5674) did not exhibit any kind of disease symptoms and their growth aid development was equivalent to control plants indicating that Bacillus subtilis ssp. shriramensis (MTCC-5674) is non-pathogenic for plant species (Fig. 18).

Example 5 5,1 Formulation of antimicrobial and/or antifungal compositions containing Bacillus sublilis ssp. shriramsnsis tMTCC-S674) cells as a biological control agent

Material * Bacillus subtilis ssp. shriratmmis (MTCC*.#sf!>74): * 'EB^hrpth. .· PPB (Potato Dextrose Broth}' * Phosphate buffer (pH 7.0) * CMC (Carboxy Methyl Cellulose)

Method 5.1.1 Preparation of; Bacillus subtílismpcs^irmneÉ^js. (MTCC-5674) cell suspension

Bacillus subiilis ssp. shrimmemis (MTGC-5674) was inoculated in 1 L sterile LB broth in a. 2 L conical flask and incubated at 30°C, for 24 hours, shaking at 200 rpm. Following the growth Bacillus subíltis ssp¿ shriramensis (MTCC-5674), the culture was centrifuged at 6,5öÖ'.fpft» M &amp; fer '1$ rain. The pellet was washed twice in phosphate buffer (pH 7.0) and piixed with 1% CMC (Carboxy Methyl Cellulose) in phosphate buffer (pH 7.0) to prepare a slurry containing 6xl07 efu/ml. The slurry •contídBÉpgiolgpl/ltty mblilis ssp, shrlramensis (MTGCk5674) was used to spray on plantsarid treat plant seediing roots by dipping. 3.1.2 To test the efficacy of formulate containing antimicrobial and/or antifungal agent to inhibit the infestation of Ehimelormi solani (NFCCI-3194) in the roots of tomato plants

Material * Slum' containing Bacillus subtilis ssp. shriramensis (MTCC-5674) « Rkizoctoma ^ip«i plFCCTMM) fungus (causes sheath blight in members of family solanacea). * Soil rite * Tomato seedlings 5.1.3 Preparation oí Rhizoctonia solani fNFCCl-31941

Rhizoctonia solani (NFCCI-3194) was grown in Potato Dextrose Broth (prepared as per the method provided in the Annexure - I (V) medium for 6 days. Following the growth of the Rhizoctonia solani (NFCCI-3194\ it was thoroughly mixed with soil rite and incubated for 15 days at room temperature. The soil rite containing the fungus was mixed with soil in 1:1 ratio. • Tomato seedlines

Tomato seedlings of 10 cm height were used in this study Method

The experiment was carried out as described below A. Tomato seedlings were planted in the soil containing Rhizoctonia solani (NFCCI-31941 but were not treated with Bacillus subtilis ssp. shriramensis (MTCC-5674).

B; The roots of Tomato seedlings were treated with slurry containing Bacillus subtilis ssp. shriramensis (MTCC-5674) and were planted in the soil containing Rhizoctonia solani (NFCCI-3194Y C. Tomato seedlings without any treatment. A. Seedline treatment with Bacillus subtilis ssp. shriramensis (MTCC-5674) cells and fungus Rhizoctonia solani fNFCCI-3194)

Tomato seedling roots were dipped in the Bacillus subtilis ssp. shriramensis (MTCC-5674) cell formulate for 30 min. The treated seedlings were planted in the pot containing soil mixed with the fungus Rhizoctonia solani (NFCCI-3194).

Control seedlings

For inducing the disease in the seedlings, tomato seedlings (untreated) were planted in the pot containing soil mixed with the fungus Rhizoctonia solani (NFCCI-31941

Tor negative control tomato seedlings (untreated) were planted in pot containing soil which is not mixed with the fungus Rhizoctonia solani fNFCCI-31941

All the pots containing tomato seedlings were transferred to the green house and maintained till fruiting stage.

Result

From the observations it was concluded that the seedlings treated with combination of Bacillus subtilis ssp. shriramensis (MTCC-5674) and the fungus Rhizoctonia solani (NFCCI-3194) grew very well equivalent to control plants, whereas the seedlings (««treated) planted in the pot eornarning fungus Rhizoctonia soktni (NFCCI-3I94) ί; exhibited retarded growth, poor flowering and fouit formation as compared with control Hence, it can beeiat'-coneluded that\Bmiiiits siiMHs ssp. skriramensis (MTCC-5674) inhibited the growth of the fungus Mímcimiü mlani (NFCCI-3194) in the rhizosphere area of torpato seedlings and protected fp seedlings from disease causing fungus (Fig. 19).

Example 6 6.1 in vitro evaluation of minimum number of Bacillus subtiiLs sap, shriramemis CMTCC-5674) cells which can control the infection of germinating corn seeds bv soil and seed borne fungal pathogen PeniciUinm oxolicitm (NFCCI-1997). 6.1.1 Preparation oí bacterial and ñinga! suspension cnltures

Materials a. Bacillus subtilis ssp. shr-immemfs b. Pen/c////nni oxfaóaou ;(|^FCCi” 1997)-Hrmt pathogenic fungus c. Carbendazim WP50 (cpmtpcroiai fopgieide} d. Luria Bertani Broth (LB)

Method i 6.1.LI Preparation of suspension culture of Bacillus subtilis ssp, shrinmensis (MTCC-5Ö74) A pure colony of .Bacillus -Mbtilis ssp- shriramensis (MTCC-5674) (Fig- 20-1) was inoculated ln 10 ml LB broth and incubated at 30^0 for 24 h at ISO rpm, For preparation of bio-control formulation l mi of fresh culture was inoepfcafldi&amp;W at 30°C for 24. h at 180. rpm. Growth, by -periodic, measurement of absorba^e^ef cu¿tói&amp;ást.:;6íif -taa. 2¾¾ bacterial cells were harvested by centrifugation and washed withcsteriie phosphate bufe, by centrifugation at 5500 rpm for 10 min at The Ceils Were finally suspended in 5ml of sterile phosphate buffer. This concentrated suspension was used for preparation of bio-con trol formulations. 6y L lii2 Preparation of suspension culture of Peniciilmm oxalicum (NFCCI-1997) iMgdUfathogen)

Pure colony of Penicillium oxalicum (NFCCI-1997) (Fig. 20-2) was inoculated on PDA plate and incubated at 28°C till spore formation. A loop full of the fungal spores were inoculated in 100 ml of PDB and incubated at 28°C for 7 days at 180 rpm. The aqueous part of the culture containing fungal spores was collected in 50 ml polypropylene tubes. The spores were washed with sterile phosphate buffer by centrifugation at 8000 rpm for 10 min at 4°C. The spores were suspended in required volume of sterile phosphate buffer to obtain a cfu of 6x104 ml"1. 6.1.2 Soil infestation with P. oxalicum fNFCCI-1997") (Tunsal pathogen')

To maintain adequate fungal spore load in the soil medium, 50 ml fungal spore suspension (6xl04 cfu/ml) was mixed with 1 kg of autoclaved soilrite and incubated for 10 days at 28°C. The soilrite colonized with fungus was uniformly mixed with soil in 1:1 ratio and filled in 96 cup trays. 6.1.3 Preparation of formulation of Bacillus subtilis ssp. shriramensis (MTCC-5674^ for biological control of soil borne plant disease

Material a. CMC (Carboxy Methyl Cellulose) b. Sucrose c. Red polymer (without fungicide) d. Bacillus subtilis ssp. shriramensis (MTCC-5674) cell suspension (bio-control agent) e. Carbendazim (commercial fungicide)

To assess effective concentration of Bacillus subtilis ssp. shriramensis (MTCC-5674) cells which can suppress growth and pathogenicity of P. oxalicum (NFCCI-1997) on germinating com seed, four different formulations were designed (details are provided in table below). Formulations containing only bio-control agent, only commercial fungicide, and one without bio-control agent or fungicide were used as controls. All the formulations contain a binding material - CMC (Carboxy Methyl Cellulose), carbon source (sucrose) and a red polymer (without fungicide). 1. Control-1 (Formulation without fungal pathogen and bio-control agent)

This formulation is composed of \% CMC, 2% sucrose, and fed polymer. This formulation has no bio-control agent, disease causing agents and fungicide. Seeds treated with this formulation were used as control seeds.

Composition

2. Control-2 (Formulation with fungal pathogen but no bio-control agent)

This formulation is composed of 1% CMC, 2% sucrose, and red polymer. It has no bio-control agent/commercial fungicide, but the seeds treated with this formulation were sown in the soil inoculated with P. oxaltcum (NFCCI-1997) fungus. As there is no biological or chemical protection around the seeds, the fungus grows profusely, infects the seeds and develops disease in the seedlings. The seeds treated with this formulation are used as diseased controls.

Composition

3. Control-3 (Formulation with commercial fungicide “Carbendazim WP50”) .

This formulation is composed of 1% CMC, 2% sucrose, red polymer and a commercial fungicide Carbendazim WP50 (trade name Bavistin) was used at a concentration of 500 pg/ml (Mohiddin et ai, 2013). This formulation is used to compare the efficacies of both bio-control agent and the commercial fiingicide in suppressing the fungal growth in the viciinity of the germinating seed.

Composition

4a. Formulation with Bacillus subtilis ssp. shriramensis (MTCC-56741 (5x104 cfu)

This formulation is composed of 1% CMC, 2% sucrose, red polymer and Bacillus subtilis ssp. shriramensis (MTCC-5674) cells at a concentration of 5xl04 cfu/ml. This formulation has minimum number of Bacillus subtilis ssp. shriramensis (MTCC-5674) cells.

Composition

4b. Formulation with Bacillus subtilis ssp. shriramensis (MTCC-5674^ cells (5x10s cfu> This formulation is composed of 1% CMC, 2% sucrose, red polymer and Bacillus subtilis ssp. shriramensis (MTCC-5674) cells at a concentration of 5x10s cfu/ml. Composition

4c. formulation with Bacillus subí ¡Us ss ip, xhriramemis (MTCC-5674) cells (5x10s cM Thi§· -coan|3M&amp;&amp;ed of 1% CMC.. 2¾ sucrose, red polymer and Bacillus (MTCC-5674) cells ata concerUption of 5x 10*cm/ml.

Composition

4d. Formulation comaxmm Bacillm subtilis shriramemis ceils (5x10? etui

This tomndation is composed of 1% CMC, 25¾ sucrose, red polymer .and. Bacillus $uMU$Mp. shrtmtmmls {MT€C~5674} cells at a concentration of 5xi0' cfti/tni (50 millioa celis/mi of carrier). This: ;ß>itpul^bh' has maximum number of Bacillus: subtilis ssp, skriramemis (MIGC-S67Í) cells.

Composition

This formulation is composed of 1% CMC, 2% sucrose, red polymer and Bacillus subiUis ssp, skriramemis (MTCC~5674) cells at a concentration of 5x10'' cfu/ml, This ihrmtdation has maxinutm number of Bacillus subtills · ssp. shriramensis(MTCC-5674) cells and is used to study the effect of biocontrol agent on seed germination and plant growth.

Composition

Table-4 Experiment plan in tabular form

*1 (Control-1)- Formulation without fungal pathogen and antifungal agent; *2(Control-2)- Formulation with fungal pathogen but no bio-control agent; *3(Control-3)- Formulation with commercial fungicide “Carbendazim WP50”; *4a- Formulation with Bacillus subtilis ssp. shriramensis (MTCC-5674) (5x104 cfu); *4b- Formulation with Bacillus subtilis ssp. shriramensis (MTCC-5674) cells (5x105 cfu); *4c- Formulation with Bacillus subtilis ssp. shriramensis(MTCC-5674) cells (5xlOácfu) ; *4d- Formulation containing Bacillus subtilis ssp. shriramensis (MTCC-5674) cells (5xl07 cfu) and *5- Formulation with only Bacillus subtilis ssp. shriramensis (MTCC-5674) cells (5xl07 cfu).

Seed coating

The bio-control formulations as per the compositions given above were coated on com seeds (Fig. 21). Twenty com seeds in triplicates (total 60 seeds) for each treatment were surface sterilized with 0.1% HgCh for 10 min and rinsed with 95% ethanol, and washed with sterilized water for 10 min each. Dry seeds were coated with 171 μ1/60 seeds of different formulations and air dried for 2 h.

Seed sowing

All the treated seeds were sown in 96 cup trays with three replicates per treatment. All trays were kept in glasshouse and maintained under controlled conditions. From seed germination onwards the trays were monitored till 5 weeks.

Data Recording Germination percentage

Germination percentage of all the seed treatments was recorded after 1 week of seed sowing.

Disease Incidence

Disease incidence was recorded as percentage after 4 weeks of seed sowing. The formula used for recording disease incidence (Hoffman et al, 2002) is as follows:

Results

Seed germination and seedling survival

Optimum seed germination i.e., 93.33%, 96.66%,100.00%, 100.00%, 100.00% and 100.00% was recorded in seeds treated with formulations - 3(control-3), 4c, l(control-l), 4b, 4d and 5, respectively, followed by 83.33% 33.33% in the seeds treated with formulations - 4a and 2(control-2). The seedling survival rate after 4 weeks of sowing was recorded as 100.00 % in the seeds treated with all the formulations mentioned above except in the seeds treated with the formulation-3 (which has commercial fungicide), this clearly indicates that the commercial fungicide “Carbendazim WP50”, though it was efficient in suppressing fungal growth, but was not 100.00 % efficient. The formulations containing different concentrations (except formulation 4a- which hás least number of cells) of Bacillus subtilis ssp. shríramensis (MTCC-5674) cells proved to be 100.00 % effective in protecting seeds from P. oxalicum (NFCCI-1997) present in the soil.

The drop in germination rate of seeds treated with formulation - 4a (formulation with least concentration of bacterial cells, 50,000 cells/ml carrier) is clear indication that a basal dose of bacterial cells is required to confer protection to the germinating seeds against P. oxalicum (NFCCI-1997) present in the soil. Thus, formulation-4b, which has a bacterial concentration of 5x10s cfu/ml (0.5 million cells/ml) conferred good protection against P. oxalicum (NFCCI-1997) and gave 100% seed germination and seedling survival rate, same as control seeds.

Table-5 Detection of effective concentration (cfu/g carrier) of Bacillus subtilis ssp. shriramensis (MTCC-5674) cells for suppressing P. oxalicum (NFCCI-1997) growth and pathogenicity.

* 1- Com seed +· red polymer -f CMC + Sucrose; *2* Com seed red polymer -r CMC + Sucrose f P, malmm (NFCCbl 997); *5~ (.lorn seed + red polymer*· CMC + Sucrose ·(· P. oxaficum (NFCC1-1997') ·* Carbendazlm.; *4&amp; to Ad - Corn seed + red polymer*· CMC + Sucrose + P. oxalic am (NFCCI-1997) 4- differentconcentrations of Bacillus subtilis ssp. shriramemis (MTCC. 5674} cells; *5- Cora seed *· red polymer CMC' -»· Sucrose -i- Bacillus subi'Atii ssp shriramemis (M.TCC-5674).

Disease incidence

The results of the study showed that there was significant difference between the treatments. The seeds treated with the formulations 1, 4b, 4c, 4d and 5 did not exhibit any disease symptoms and displayed healthy growth, similar to control seedlings, indicating that the biocontrol agent Bacillus subtilis ssp. shriramensis (MTCC-5674) present in the formulations greatly suppressed growth and pathogenicity of the fungus P. oxalicum (NFCCI-1997), and thus protected the seeds from getting infected with the fungus. The seeds treated with the formulation-3 (which has a commercial fungicide Carbendazim 50WP) showed a diseáse incidence of 3.57 % indicating that though the commercial fungicide was effective in suppressing the fungal growth, but not as good as bio-control agent used in this study. Table-6 Percentage disease Incidence of com seedlings treated with different formulations.

Disease incidence of 82 % and 100 % was recorded in the seeds treated with the formulations 4a and 2, respectively. The results indicate that the Bacillus subtilis ssp. shriramensis (MTCC-5674) cell density present in the formulation 4a was not effective in suppressing growth of fungus and hence the germinating seeds were infected with the fungus and died after 2 weeks of germination. As expected, the seeds treated with formulation -2 which has neither bio-control agent nor commercial fungicide showed 100% \ disease incidence indicating that the fungus infected the germinating seeds and killed the seedlings within 2 weeks of germination.

Conclusion

The above results clearly indicate that Bacillus subtilis ssp. shriramensis (MTCC-5674) at a concentration of 5x10s cfu/ml (formulation-4b) is effective in suppressing growth of fungal pathogen and gives 100% protection to germinating seedlings of com. Hence, the bio-control agent can be successfully used in coating seeds for effective control of soil borne pathogenic fungus P. oxalicum (NFCCI-1997). 6.2 Testing the efficacy of the Bacillus subtilis ssp. shriramensis (MTCC-5674") cells formulation in promoting the growth and yield in plants

Materials and methods Materials 1. Seeds of Com, Tomato and Brinjal treated with the formulation as mentioned in 6.1.3 (4b). 2. Control seeds of Com, Tomato and Brinjal.

Methods

Seed coating

Seeds of Com, Tomato and Brinjal were treated with the formulation mentioned in 6.1.3 (4b) and air dried.

Seed sowing

Treated and untreated (control) seeds of Com, Tomato and Brinjal, each in three replicates and each replicate containing 23 seeds were sown in the field of 4 meters area. The standard spacing measurements like 20 cm plant to plant and 60 cm row to row distance were maintained. Appropriate agronomy practices were followed to grow diese crops to maturity.

Results

There were significant increase in plant growth parameters and yield under field conditions. The seeds coated with formulate containing Bacillus subtilis ssp. shriramensis (MTCC-5674) cells increased the yield in Com, Brinjal and Tomato by 17.60, 37.15 and 1.58%, respectively (Table -7). The Com, Brinjal and Tomato plants showed higher rate of growth, development and biomass accumulation (a representative picture of difference in treated and untreated Com is given in Fig 25). Earlier reports on plant growth promoters have also proved that the formulation containing Bacillus subtilis enhanced the growth of plants and induced systemic resistance to disease protection by producing 60 different types of secondary metabolites (Compant et ai, 2005 and Mohan Kumar et al., 2015).

Table- 7 Effect of Bacillus subtilis ssp. shriramensis (MTCC-5674) cells formulation on the yield of Com, Tomato and Brinjal.

Example 7 7.1 Screening the efficacy of antifungal/antimicrobial agent to inhibit the growth of human pathogenic fungi A range of fungal species causing diseases in human beings were isolated from the people suffering from various skin and lung infections. The antifungal and/or antimicrobial activity was tested against all the isolated human pathogenic fungi. Materials and Methods Materials 1. Penicillium ssp. 2. Aspergillus flavus < 3. Aspergillus niger 4. Aspergillus nidulans 5. PDA plates 6. Antifungal/antimicrobial agent isolated from Bacillus subtilis ssp. shriramensis (MTCC-5674)

Method

An aliquot of 500 μΐ of the culture filtrate containing antimicrobial and/or antifungal agent was added into the wells made in the PDA agar and a loop full of test fungi were inoculated at the other comer of the respective plates labeled with the respective fungus and incubated at 28°C till the growth of fungal mycelium was observed in the vicinity of the well containing culture filtrate.

The inhibitory activity of the filtrate against the target fungus was recorded in millimetres as the inhibitory zone formed surrounding the well.

Result A range of fungal species causing diseases in human beings were isolated from the people suffering from various skin and lung infections tested in the antimicrobial and/or antifungal assay and all of them demonstrated complete inhibition of growth in the presence of Bacillus subtilis ssp. shriramensis (MTCC-5674) culture filtrate (Fig. 24). Conclusion

From the observations it has been concluded that the Antifungal/antimicrobial agent isolated from Bacillus subtilis ssp. shriramensis (MTCC-5674) can be used in pharmaceutical applications also.

References 1. Compant , S., Duffy, B., Nowak, J., Clement, C. and Barka, E.A. (2005). Use of plants growth promoting bacteria for biocontrol of plant diseases: Principles, Mechanisms of action, and future prospects. Appl. Environ. Microbiol. 71:4951-4959. Sei. World J., Vol 2012, pp. 001-012. 2. Hoffmann, W.A. and Poorter, H. 2002. Avoiding Bias in Calculations of Relative Growth Rate. Ann. Bot., Vol.90 (1), pp. 37-42. 3. Li, J. Yang, Q. Zhao, L-H, Zhang, S.M., Wang, Y.X. Xiao-yu and Zhao, X.Y. 2009. Purification and characterization of a novel antifungal protein from Bacillus subtilis strain B29. J. Zhejiang Univ. Sei. B.,Vol. 10 (4) pp. 264-272. 4. Malusá, E. Sas-Paszt, L. and Ciesielska, J. 2012. Mena-Violante, H.G. and Olalde-Portugal, V. 2007. Alteration of tomato fruit quality by root inoculation with plant growth-promoting rhizobacteria (PGPR): Bacillus subtilis. BEB-13bs. Sei. Hort.,Vol.l (113), pp. 103-106. 6. Mohan Kumar, S.P., Chowdappa, P. and Krishna, V. (2015). Development of seed coating formulation using consortium of Bacillus subtilis OTPB1 and Trichoderma harzianum OTPB3 for plant growth promotion and induction of systemic resistance in field and horticultural crops. Indian Phytopath. 68 (1):25-31. 7. Mohiddin, F. A. and Khan, M. R. 2013. Tolerance of fungal and bacterial bio-control agents to six pesticides commonly used in the control of soil borne plant pathogens. Global J. Pests, Dis. Crop Prot., Vol. 1 (1), pp. 001-004.

Relevant Patents 1. A novel strain of Bacillus for controlling plant diseases and com rootwonn. (EP981540A1). 2. Strain of Bacillus subtilis for agricultural use. (W02009031874A1). 3. Antifungal Bacillus subtilis and a microorganism wettable powder containing the same (KR2011075132A). ámmm&amp;l

Cm!?osMionoíCuSmr«MediiyusM

Note; A general method of media preparation is provided below. All the media compositions given below are for 100 ml volume. Compositions ehanges depending upon the quantity required, {i) Emparatlomolll br^_(pjl-dj}

Composition ofT3 medium

Meüisl

All the medía componente were weighed and taken in a glass bottle and dissolved in distilled water. The container with the medium was autoclaved at 12· PC for 15 min. (11) Premtio^

Composition of T3 medium

Method

Ail the media components were weighed and taken in a glass bottle and dissolved in distilled water. The glass bottle with the medium was autoclaved at 12PC for 15 min. After autoclaving, the medium was poured in to sterile peiri plates £111) PrepkäS^

Composition of LB medium llÉÉísá

All the media components were weighed and taken in a glass bottle and dissolved in distilled water. The container with the medium was aufoeiaveefat:l:Í:t^i,íbr 15 min. (IV) Prenatation ot.LB agar platgg.Cplinl?.^.

Composition of LB medium

Method

All the media components were weighed; and taken hi a glass bottle and dissolved in distilled waten The glass bottle with the ntedkan was autoelaved at 121¾ for 15 min. Atter autoelavm^^e--medim:MS poured in tosteniepetri plates, (VI Preparation ofPDB broth ipH - 6.0)

Composition of PDB medium

Method

Potato powder (2.0 g} was weighed and taken in a 250 mTglass bottle containing 50 ml of distilled: water and boiled for 5 min. The boiled potato water was .filtered using muslin cloth. The filtrate was collected in a itesh 2SÖ ml glass bottle and 2.0 g of dextrose -was added to it. Alter making the total volume to 100 ml, the bottle with the medium was autoclaved at 121°C for IS min. f ¥if retMrati.öft of PDA agar plates foil mJhii)

Composition of PDA medium

Method

Potato powder (2,0 g) Was weighed and taken in a 250 mi glass bottle containing 50 ml of distilled water and foiled for 5 min. The boiled potato water was filtered using muslin cloth. The filtrate was collected ;|n a fresh 25Ö ml glass bottleand2.0 g of dextrose and 2.0 g of agar were added to it. After making the total volume to 100 ml, the bottle with the medium was autoclaved at 121 ÖC for 15 mim The molten PDA was poured into the sterile petri plates. (VIII Preparation of Hush and Leifeon OP BáMlMedlom (OFBM^ (pB ~ 7,1) Composition of OFBM medium

I \ i j

Method

All the; media components ryere weighed and taken in a glass bottle and dissolved in distilled water. The glass bottle with the medium was autoclaved at 121°C for 15 min. After •atttoelavihg* the medium wss-:pp«f©d 'fold sterile pohtsre pibes* (¥11 Preparation of SIM f Sulphide Indole Motility) medium (PR ~ 7.Ti Composition of SIM medium

Method - AD the media components were weighed and taken in a glass bottle arid dissolved in distilled water. The glass bottle with the medium was autoclaved at 12PC for 15 rain. After autoclaving, the medium of molten stage was poured in to sterile culture tubes.

Annexurc II

Preparation phosphate buffer

Method

Both the phosphate salts were taken in a glass beaker, SO ml distilled water was added to salts and stirred on a magnetic stirrer using a magnetic bar. After ensuring that the phosphate salts are completely dissolved, the solution was made up to 100 ml with distilled water.

Annexlire III

Carriers and other agents used in the experiment

Abbreviations

Claims (14)

1. We Claim:

   1. A novel bacterium belonging to Bacillus species exhibiting antimicrobial and/or antifungal and plant growth promoting activity.
2. 2. The novel bacterium as claimed in claim 1, wherein the novel bacterium is isolated Bacillus subtilis ssp. shriramensis having the accession number (MTCC-5674).
3. 3. An extract of the novel bacterium belonging to Bacillus species as claimed in claim 1, exhibiting antimicrobial and/or antifungal activity.
4. 4. A pure culture of the novel bacterium as claimed in claims 1 and 2.
5. 5. The extract as claimed in claim 3, wherein the process of its production comprises a. Growing the Bacillus subtilis ssp. shriramensis having the accession number (MTCC-5674) in a T3 medium having pH 6.8 in a shaking incubator at 30°C for 60 h; b. Recovering the extract having antimicrobial and/or antifungal activity
6. 6. The process as claimed in claim 5, wherein the Bacillus subtilis ssp. shriramensis is grown under aerobic conditions.
7. 7. The process as claimed in claim 5 optionally comprises concentrating the extract using conventional methods.
8. 8. A composition comprising the novel bacterium as claimed in claim 1, wherein the composition has antimicrobial and/or antifungal and plant growth promoting activity at a concentration 5x10s cfu/ml to 5xl07 cfu/ml of the bacterium.
9. 9. A composition comprising the extract as claimed in claim 3, wherein the composition has antimicrobial and/or antifungal activity at a concentration of 4 pg/pl'to 20 pg/|il of the extract.·
10. 10. A composition comprising the novel bacterium as claimed in claim 1 and the extract as claimed in claim 3, wherein the composition has antimicrobial and/or antifungal and plant growth promoting activity.
11. 11. The composition as claimed in any of the preceding claims optionally comprises one or more antimicrobial and/or antifungal and plant growth promoting agents.
12. 12. The composition as claimed in any of the preceding claims optionally comprises agriculturally acceptable carrier.
13. 13. A method for inhibiting growth of pathogenic fungi and/or bacteria, wherein said method comprises contacting the pathogenic fungi and/or bacteria with an effective amount of 5x10s cfu/ml to 5xl07 cfu/ml of the novel bacterium as claimed in claim 1 or the composition as claimed in any of the preceding claims.
14. 14. The novel bacterium or the extract as claimed in claims 1 and 3 or the composition as claimed in any of the preceding claims whenever used for the preparation of an antimicrobial and/or antifungal and plant growth promotion composition for inhibiting the growth of pathogenic fungi and/or bacteria.